Combinatorics

Pathology

Mathematics

Biology

Antigen

Enumeration

Antibody

Immunology

Immunofluorescence

Computational biology

Medicine

Immune system

Nature Protocols

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging